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ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13621/m_MTBLS13621_LC-MS_positive_reverse-phase_metabolite_profiling_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13621/m_MTBLS13621_LC-MS_positive_reverse-phase_metabolite_profiling_v2_maf .tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13621/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13621/s_MTBLS13621.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13621/a_MTBLS13621_LC-MS_positive_reverse-phase_metabolite_profiling.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13621/FILES/DERIVED_FILES/Two-step_pos1.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13621/FILES/DERIVED_FILES/LB_pos3.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13621/FILES/DERIVED_FILES/LB_pos2.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13621/FILES/DERIVED_FILES/Two-step_pos3.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13621/FILES/DERIVED_FILES/Two-step_pos2.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13621/FILES/DERIVED_FILES/LB_pos1.mzMLprimary200OKftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13621Metabolite identification was conducted using MS-DIAL(4.9) software, leveraging high-accuracy mass measurements and diagnostic fragmentation patterns with standard mass tolerance settings. Only compounds exhibiting reproducible detection and high spectral matching were considered confidently identified. In accordance with the Metabolomics standards initiative, these metabolites were classified as Level 2. MetaboLightsPublicLiquid Chromatography MS - positive - reverse-phaseChromatographic separation was performed on a reversed-phase C18 column (Zorbax Eclipse Plus C18-Rapid Resolution HD) maintained at a controlled temperature (35°C). Mobile phase A consisted of water with 0.1% formic acid, and mobile phase B consisted of acetonitrile with the same additive.The injection volume was typically 10 μL.The LC gradient elution program was as follows: 100% solvent A at 0 min, maintained at 100% A until 2.5 min; decreased to 50% A at 7.5 min; further decreased to 0% A at 11.5 min and held at 0% A until 14 min; and finally returned to 100% A at 20 min. The total run time was 20 min. The autosampler was maintained at 4–10°C to preserve sample stability.Biosynthesis of polyamine–polyphosphate granules for colitis alleviation.Zihao FanNanjing universityMixturemass spectrometryMetabolites were extracted under cold conditions to limit enzymatic activity. Briefly, samples were weighed and mixed with pre-chilled extraction solvent (methanol:water) at a fixed ratio (e.g., 10–20 μL solvent per mg sample). The mixture was vortexed, sonicated in an ice-water bath, and incubated at −20°C to precipitate proteins. After centrifugation (15,000 × g, 10 min, 4°C), the supernatant was collected. When needed, the extract was evaporated under nitrogen or in a vacuum concentrator and reconstituted in an appropriate solvent for LC–MS analysis. Extracts were filtered (0.22 μm) prior to injection.Citrobacter freundiihttps://www.ebi.ac.uk/metabolights/MTBLS13621Zihao Fan. Nanjing university. 845640877@qq.com.Raw MS data files were converted to an open format (mzML) using vendor software or ProteoWizard/MSConvert with centroiding enabled when appropriate. Converted files were imported into MS-DIAL (4.9) for peak detection, deconvolution, alignment, and normalization using default or optimized parameters. Quality control procedures included checking retention time stability, mass accuracy, and signal consistency across runs. Only features detected reproducibly across biological replicates and meeting quality thresholds were retained for downstream identification.Cultivation parameter845640877@qq.comThe CPP strain was cultured overnight in LB medium at 37.0°C under shaking conditions to ensure uniform growth without PolyP accumulation. Cells were harvested by centrifugation at 8000 rpm for 5 min, washed twice with 20 mM PBS buffer (pH 7.0) to remove residual medium, and then resuspended in the same buffer to obtain PolyP-free cells. To assess the performance of the engineered strain, cells were cultivated in a synthetic medium (SM) devoid of chemical inducers or antibiotics. The SM was prepared with deionized water, contained the following components per liter: 100 mg tryptone, 50 mg NaCl, 226 mg MgSO4·7H2O, 180 mg NH4Cl, and 1 mg yeast extract. Additionally, KH2PO4·3H2O (102 mg/L) was added to maintain a total inorganic phosphorus (Pi) concentration of 20 mg P/L, ensuring non-limiting phosphorus condition unless otherwise stated. PolyP-free cells were inoculated into SM in 1000 mL Erlenmeyer flasks at an initial OD600 of 0.15. Cultivation (500 mL) was carried out at 37°C with shaking at 200 rpm. At specified time intervals, 4 mL aliquots were collected for analysis. Metabolomicspolyphosphatebiosynthesisuntargeted metabolitesBacteriaColitisPolyaminespolyphosphatebiosynthesisuntargeted metabolitesBacteriaColitisPolyaminesMass spectrometric detection was carried out using a AGILENT 6545 QTOF equipped with an electrospray ionization (ESI) source operated in both positive and negative ion modes. Typical source settings included a capillary/spray voltage ( 5.5 kV), Full-scan MS spectra were acquired over an appropriate m/z range (50–1,000). MS/MS spectra were collected in data-dependent acquisition (DDA) mode using predefined collision energy settings and an isolation window suitable for structural elucidation. Lock-mass or internal calibration was applied when available to ensure mass accuracy.falseBiosynthesis of polyamine–polyphosphate granules for colitis alleviation Natural polyamines, such as spermidine and spermine, exert strong anti-inflammatory effects, but their therapeutic benefits are limited by rapid absorption in the small intestine, resulting in low colonic availability. In this study, engineered Citrobacter freundii overexpressing ppk1 (CPP) could significantly enhance both intracellular polyamine synthesis and polyphosphates (PolyP) accumulation. Under a two-step cultivation process, the engineered strain produced substantial amounts of polyamines, including spermidine (145.14 ± 5.11 mg/g) and spermine (175.31 ± 4.2 mg/g). CPP responds to environmental stress by importing large amounts of phosphate for PolyP synthesis. This process further promotes polyamine production, which binds to PolyP to neutralize intracellular charge. The resulting polyamine–polyphosphate granules (PPGs) enhance both binding stability and slow-release properties in vivo. Notably, oral administration of PPGs increased colonic polyamine levels, alleviated DSS-induced colitis, reduced inflammatory cytokines, and restored gut microbiota balance. Overall, biosynthesized PPGs represent a highly promising material for mitigating colitis.2026-01-072026-01-07MTBLS13621